Diagnostic, prognostic, therapeutic and screening protocols

ABSTRACT

The specification describes an antibody capture process comprising (i) obtaining a biological sample comprising antibodies, (ii) contacting the biological sample with recombinant pIgR or a dIgA-binding variant, wherein the pIgR or variant binds dIgA and forms a pIgR-dIgA complex. The process may further comprise (iii) directly or indirectly assessing the level of the pIgR-dIgA complex or the level of a complex between pIgR-dIgA and an antigen of interest. There is also an antibody capture process for determining gut wall integrity in a test subject, wherein the level or ratio of SIgA to dIgA is compared to a corresponding level or ratio from a control subject. The specification provides kits embodying the process and recombinant pIgR when used for, or for use, in capturing or detecting dIgA and/or IgM.

PRIORITY CLAIM

The present application claims priority from Australia ProvisionalPatent Application No. 2012904887 filed on 8 Nov. 2012, the disclosureof which is included herein by reference.

FIELD

The present specification relates generally to the fields of diagnostic,prognostic and therapeutic protocols with respect to infectious agentsor other conditions associated with immune activation and particularlymucosal immune activation. More particularly, the specification relatesto the use of antibodies as biomarkers for immune activation and/or fordiagnosis, prognosis and treatment of conditions associated with immuneactivation. The present protocols are proposed for ready translationinto both laboratory and point of care formats to reach targetpopulations worldwide.

BACKGROUND

Bibliographic details references referred to in this specification arelisted at the end of the specification.

The reference to any prior art is not and should not be taken as anacknowledgment or any form of suggestion that this prior art forms partof the common general knowledge in any country.

The detection of specific antibody (immunoglobulin (Ig)) classes isrecognized as an important step in diagnostic and research methods forhuman and animal diseases. For example, detection of antigen-specificIgM-class antibodies is widely used as a diagnostic test for infectionwith viruses such as hepatitis A virus, hepatitis E virus, West Nilevirus, dengue viruses, measles virus, rubella virus; and for infectionwith bacteria such as syphilis (Treponema pallidum), because IgM classantibodies are typically made in the body of an infected host during theacute phase of infection and are detectable for only a few months.

Conversely, IgG-class antibodies commonly persist for life and mayindicate either current or past infection with a specific agent. Forchronic infections such as the human immunodeficiency virus (HIV) wherepatients do not spontaneously clear the virus, detection of IgG-classantibodies is diagnostic for infection, whereas for others such ashepatitis C virus (HCV) where a proportion of patients do clear thevirus either spontaneously or following treatment, the detection ofantigen-specific IgG is not diagnostic of current or ongoing infection.IgG-class antibodies are also primarily responsible forantibody-mediated immunity within the plasma compartment of the body.

IgA-class antibodies have also been used to aid diagnosis of infectionsincluding hepatitis E virus, hepatitis A virus, and dengue viruses, aswell as in the study of vaccines and immunity to infections. IgA isattractive for diagnostic purposes, because it is predominantly madeduring the acute phase of infection, and high levels of antigen-specificIgA can provide a marker of current infection, with or without theconcurrent detection of IgM. In addition, because IgA is the predominantantibody class that is secreted at mucosal epithelial surfaces, itspresence is considered as a marker of mucosal immunity. The role ofdifferent IgA structural forms as biomarkers for infection, such asspecifically dIgA, is not understood. The role of SIgA in infection andantigens that engender SIgA responses have not been explored nor havediagnostic and prognostic protocols been developed that are designed torapidly and conveniently assess these responses in patient sera.

In most animals, IgA is synthesized almost exclusively as dimeric orhigher polymeric forms, here described collectively as dIgA, which areable to interact with the polymeric Ig receptor (pIgR). This interactionresults in secretion of large amounts of secretory IgA (SIgA) into thelumen of epithelial tissues (see FIGS. 1 and 2). However, in humans andhigher primates the dIgA is only a minor fraction of the total IgA, withmonomeric IgA (mIgA) representing around 90% of the total IgA, anddimeric or higher polymeric forms of IgA representing around 10% of thetotal IgA.

Detection of IgA, IgM, IgG and other antibody classes or isotypes isusually performed using antibody reagents prepared in another species,for example rabbit antibodies specific for human IgM, or mousemonoclonal antibodies specific for human IgA, or monoclonal antibodiesspecific for individual antibody subclasses such as IgA1, IgA2 or IgG1,IgG2a, IgG2b, IgG3, IgG4. Antibody based capture assay are associatedwith levels of non-specific binding which is minimised throughoptimisation protocols.

There is a need for improved serological protocols for monitoringinfections associated with mucosal surfaces and mucosal immune responsesin a subject, and for agents that can be used to assess dimeric orpolymeric antibody production and or for dimeric or polymeric antibodypurification.

SUMMARY OF EMBODIMENTS

In one embodiment, therefore, the specification provides an antibodycapture process comprising (i) obtaining a biological sample comprisingantibodies, (ii) contacting the biological sample with recombinant pIgRor a dIgA-binding variant, wherein the pIgR or variant binds dIgA andforms a pIgR-dIgA complex. Once the complex has been formed, the complexmay be quantified. In some embodiments, dIgA may be released from thepIgR and further processed. In one embodiment, the process is employedfor the purification of dIgA antibodies as an alternative to existingprocesses which employ jacalin agarose. Detection of complexes usesroutine methods and agents known in the art such as ELISA or otherimmunoassay based methods.

In one embodiment, the process further comprises (iii) directly orindirectly assessing the level of the pIgR-sIgA complex or the level ofa complex between pIgR-dIgA and an antigen of interest.

In one embodiment, the pIgR or a dIgA-binding variant binds dIgA andsubstantially fails to bind IgM or wherein the pIgR or variant bindsdIgA and IgM. Processes for detecting antigen specific IgM and dIgA maybe used in conjunction with tests for total IgA, IgG and otherindividual isotypes, subclasses and structural forms and combinations oftwo or more of these.

In one embodiment, the biological sample is a blood or serum sample.Alternative biological samples include samples comprising cellsexpressing dIgA. Thus, in some embodiments, the process may be used todetect individual B-cells that express dIgA in the screening orisolation of immortalized B-cells.

In another embodiment, the biological sample is obtained from a subject.The subject is human or primate in some embodiments, and a mammalian oravian animal species other than a primate or human species in anotherembodiment. Various forms of recombinant pIgR are selected based on thetarget antibody of interest and species from which the antibody isderived.

As illustrated herein in one embodiment, the pIgR is recombinant HpIgAor RpIgR. conveniently, in one embodiment, the recombinant pIgR ordIgA-binding variant has the transmembrane domain and/or the cytoplasmicdomain deleted. In other embodiments, the recombinant pIgR comprises aheterologous detection or binding domain. In another embodiment, therecombinant pIgR or IgA-binding variant is recombinantly produced in aglycan deficient cell, such as a CHO cell.

In exemplary embodiments of the process, the recombinant pIgR is boundto a solid support.

In one embodiment, the biological sample is depleted of IgM or dIgAantibodies prior to use in the process. this allows the pIgR which bindsto IgM and dIgA to be employed in assays to specifically detect dIgA.Similarly, depletion of dIgA (using, for example, R/HpIgR) facilitatesthe use of HpIgR in assays to specifically detect IgM. In someembodiments, IgG is depleted prior to sample use in order to reducecompetition with dIgA. However, as competing antibodies are washed awayin the present antibody/isotype capture formats, lack of suchcompetition is an advantage of the process.

Any antigen of interest may be employed and in one non-limitingembodiments, the antigen of interest is an antigen of an infectiousagent or an antigen associated with a condition of a subject thataffects a mucosal surface or associated tissues. For example, infectiveagents include HIV, leprosy, syphilis, hepatitis, dengue virus, measlesand rubella.

In one embodiment, the process further comprises contacting thebiological sample with an anti-SC binding agent or anti-SC antibodywherein the anti-SC binding agent or anti-SC antibody binds SIgA andforms an SIgA-binding agent/antibody complex. In another embodiment, theprocess further comprises contacting a sample comprising the pIgR-dIgAcomplex with a denaturing solution to remove any SIgA from the complexand measuring the ratio of SIgA and dIgA in the biological sample.

In another aspect, the specification enables an antibody capture processfor determining gut wall integrity in a test subject, the processcomprising (i) obtaining a biological sample comprising antibodies fromthe test subject, (ii) contacting the biological sample with recombinantpIgR or a dIgA-binding variant, wherein the pIgR or variant binds dIgAand forms a pIgR-dIgA complex, and (iii) contacting the biologicalsample with a specific anti-SIgA binding agent or anti-SC bindingagent/antibody wherein the anti-SIgA binding agent or anti-SC bindingagent/antibody binds SIgA and forms an SIgA-binding agent/antibodycomplex, and (iv) measuring and comparing the level of the complexformed in (ii) with the level of the complex formed in (iii), whereinthe ratio of SIgA to dIgA is compared to a corresponding level or ratiofrom a control subject and provides a measure of gut integrity/leakage.

In one embodiment of the process, the level or ratio of SIgA2/dIgA2and/or SIgA1/dIgA1 are determined.

In another embodiment, the antibody capture process comprises (i)obtaining a biological sample comprising antibodies, (ii) contacting thebiological sample with recombinant pIgR or a dIgA or IgM-bindingvariant, wherein the pIgR or variant binds IgM and/or IgA and forms apIgR-IgM and/or pIgR-dIgA complex.

Alternatively, the antibody capture process may comprise (i) obtaining abiological sample comprising antibodies, (ii) contacting the biologicalsample with recombinant pIgR wherein the pIgR or variant binds IgM andforms a pIgR-IgM complex.

In another illustrated embodiment, a process is enabled for detectingthe presence of antigen-specific dIgA in a subject, the processcomprising (i) obtaining a biological sample comprising antibodies froma subject, (ii) contacting the sample with R/HpIgR and antigen and (iii)measuring the level of antigen-specific dIgA.

In another embodiment, a process is described for detecting the presenceof antigen-specific IgM in a subject, the process comprising (i)obtaining a biological sample comprising antibodies from a subject, (ii)contacting the sample with a HpIgR and antigen and (iii) measuring thelevel of antigen-specific IgM.

In one embodiment, the process is for detecting the presence ofantigen-specific IgM and dIgA in a subject, the process comprising (i)obtaining a biological sample comprising antibodies from a subject, (ii)contacting the sample with HpIgR and R/HpIgR and antigen and (iii)measuring the level of antigen-specific IgM and antigen specific dIgA.

In yet another aspect, the present specification enables a kit forassessing immune status in a subject, the kit comprising, (a) animmunographic device comprising a porous membrane operably connected toa sample portion, a test portion, and optionally a control portion; andfurther comprising a sucker portion, portion comprising a recombinantpIgR molecule or dIgA-binding variant thereof, a portion comprising anantigen of interest and optionally a conjugate portion; and b)instructions for using the immunographic device to detect the presenceof antigen specific dIgA antibody in a biological sample obtained fromthe subject.

As described herein in relation to the process, in some embodiments ofthe kit, the pIgR is HpIgR and/or R/HpIgR. In some embodiments, therecombinant pIgR or dIgA-binding variant has the transmembrane domainand/or the cytoplasmic domain deleted. In one embodiment, therecombinant pIgR comprises a heterologous detection or binding domain.In yet another embodiment, the recombinant pIgR or IgA-binding variantis recombinantly produced in a glycan deficient cell.

In one embodiment, the recombinant pIgR is bound to a solid support.

In one embodiment, the biological sample is depleted of IgM or dIgAantibodies prior to use in the process. The kits and reagents containedtherein of the present invention are for use ex vivo.

Any antigen may be employed however in one embodiment, the antigen ofinterest is an antigen of an infectious agent or an antigen associatedwith a condition of a subject that affects a mucosal surface orassociated tissues. Illustrative infectious agents are selected fromHIV, leprosy, syphilis, hepatitis, dengue virus, measles and rubella.The detection of elevated levels of antigen specific dIgA recombinantpIgR relative to control levels facilitates diagnosis and the selectionand treatment options. In some embodiments, a method of treatment iscontemplated comprising requesting a test for antigen-specific dIgAlevels and administered treatment to the diagnosed subject if the testis positive for an infection or condition.

In another embodiment, the kit further comprises an anti-SIgA bindingagent/antibody or anti-SC antibody, wherein the anti-SIgA bindingagent/antibody or anti-SC antibody binds SIgA and forms an SIgA-bindingagent/antibody complex.

In another aspect, recombinant pIgR is provided which is suitable foruse in capturing or detecting dIgA and/or IgM. Illustrative recombinantpIgRs include R/HpIgR or HpIgR or a dIgA and/or IgM binding variant ofR/HpIgR or HpIgR. Illustrative amino acid and nucleotide sequences areset out in SEQ ID NO:1 to 20, bearing in mind that some of thesesequence encode or provide a CD4 cytoplasmic domain which is entirelyoptional and may be deleted, modified, supplemented or replaced withother binding or detection molecules known in the art. Once the subjectinvention is contemplated, useful variants of recombinant pIgR will beapparent to the skilled person and readily made and tested.

DETAILED DESCRIPTION OF THE FIGURES SUPPORTING AND DESCRIBING THESUBJECT PROCESSES AND KITS

If figures contain colour representations or entities, coloured versionsof the figures are available from the Patentee upon request or from anappropriate Patent Office. A fee may be imposed if obtained from aPatent Office.

FIG. 1 provides a schematic of the production of dIgA and its secretionat mucosal surfaces as SIgA. Most dIgA produced in the submucosaltissues is subsequently bound to pIgR and transcytosed to the mucosalsurface, where pIgR is cleaved to produce SIgA (or free SC), with SIgAconstituting a first layer of defense against pathogens. pIgR binding isdependent on the presence of J-chain in polymeric Ig, and binding occursto both IgM and dIgA in humans.

FIG. 2 provides a representation of the structure of dIgA1 and itsinteraction with pIgR which is then cleaved to give SIgA1. Theassociated Secretory component portion of pIgR interacts with bothJ-chain and the Fc domains of both individual IgA molecules.

FIG. 3 provides a representation of the structure of chimeric R/HpIgRrelative to human (HpIgR), and the R/HpIgR that is fused to thecytoplasmic domain of human CD4 at its C-terminus. The R/HpIgR and otherforms are expressed and secreted at high levels in 293T cells, shown bythe detection of R/HpIgR using coomassie brilliant blue staining ofSDS-PAGE gel of the crude supernatant (SN) from transiently transfectedcells. R/HpIgR-cyto is readily purified to homogeneity and highconcentration by affinity chromatography with an immobilised matrix ofmonoclonal antibody 4B4 directed against the cytoplasmic domain of CD4(Pure). Either pure pIgR or crude SN can be used in detection or bindingof dIgA as preferred.

FIG. 4 provides a schematic representation of the structure offull-length (native) pIgR (TOP), relative to the recombinant formHpIgR-cyto (bottom), in which the transmembrane domain (TM) andcytoplasmic domain (cyto) of pIgR have been replaced with thecytoplasmic domain of human CD4. Because of the deletion of the TMdomain, the product is secreted from cells rather than being retained atthe cell surface.

FIG. 5 provides a schematic of the structure of full-length (native)rabbit pIgR (TOP), relative to the recombinant form RpIgR-cyto (bottom),in which the transmembrane domain (TM) and cytoplasmic domain (cyto) ofrabbit pIgR have been replaced with the cytoplasmic domain of human CD4.Because of the deletion of the TM domain, the product is secreted fromcells rather than being retained at the cell surface.

FIG. 6 provides a schematic of the structure HpIgR cyto, RpIgR-cyto, andchimeric R/HpIgR-cyto. These forms of pIgR demonstrate highly efficientbinding to solid surfaces such as polystyrene ELISA plates (Nunc Immulonor similar) through interaction of the CD4 cyto domain with the plasticor other solid surfaces.

FIG. 7 provides a schematic of the structure RpIgR and chimeric R/HpIgR.In the absence of the CD4 cyto domain, the RpIgR can be detected byreactivity with antibodies against the rabbit pIgR, and the chimeraR/HpIgR can be detected by reactivity with antibodies against the rabbit(domain 1) and/or human (domain 2-5) pIgR. The preferred antibodies mustbe able to interact with pIgR when it is bound to dIgA, not only to freepIgR.

FIG. 8 provides the results of ELISA comparing the binding of HpIgR andR/HpIgR to human IgM and dIgA. HpIgR or R/HpIgR were immobilised on96-well Nunc Immulon plates overnight at 4° C. Dilutions of purifiedhuman IgM or dIgA in PBS were bound to the immobilised pIgR formsovernight. After washing, the captured IgM or dIgA were detected usinganti-IgM or anti-IgA conjugated to horseradish peroxidase (HRP) andcolorimetric substrate TMB. The results demonstrate that HpIgR showspreferential binding to IgM (magenta) as well as binding to dIgA(green), whereas R/HpIgR shows greatly reduced binding to IgM (yellow)but retains strong binding to dIgA (blue).

FIG. 9 provides the results of ELISA comparing the detection ofimmobilised dIgA using R/HpIgR. Dilutions of purified dIgA or no dIgA(mock) were immobilised on 96-well Nunc Immulon plates that werepreviously coated with anti-IgA, so that the dIgA was bound to the plateby antibody-antigen interaction rather than passive absorption. The dIgAwas detected using R/HpIgR (“tailless”) or no pIgR (“mock”),anti-secretory component and anti-mouse HRP and TMB substrate. Theresults demonstrate R/HpIgR is able to detect dIgA at the lowestconcentration tested (31 ng/ml) with strong signal in ELISA withnegligible background.

FIG. 10 is a schematic of one preferred experimental approach fordetecting the presence of antigen-specific dIgA in a sample such ashuman serum or plasma. Recombinant R/HpIgR-cyto is immobilised on theELISA plate, and incubated with serum or other samples. Dimeric IgA iscaptured on the solid phase, and after washing to remove other samplecomponents (such as IgA and IgG that are not captured, left), thepresence of antigen-specific dIgA is detected by sequential addition ofantigen that is either biotinylated, or reacted with a biotinylatedmonoclonal antibody against the antigen, and streptavidin-HRP. In thisway, any antigen that is immobilised by reaction with antigen-specificdIgA will give a signal through the biotin-streptavidin interaction.

FIG. 11 is a schematic of one preferred experimental approach fordetecting the presence of hepatitis A virus-specific dIgA in serum(right), compared to detection of HAV-specific IgM using the standardmethod of anti-IgM capture (left).

FIG. 12 provides the results of ELISA demonstrating the detection ofHAV-specific IgM in IgM capture, using serial dilutions of serum from apatient with acute HAV infection (Accurun HAV panel sample 121). Theserum sample is either untouched before dilution (untouched, purple) orsubstantially depleted of IgM using Capture-Select IgM (BAC) (red). Theresults show that this IgM depletion method reduces the level ofHAV-specific IgM in the sample by around 256-fold compared to untouchedserum.

FIG. 13 provides the results of ELISA demonstrating the detection ofHAV-specific dIgA in R/HpIgR capture, using serial dilutions of serumfrom a patient with acute HAV infection (Accurun HAV panel sample 121).The serum sample is either untouched before dilution (untouched, purple)or substantially depleted of IgM using Capture-Select IgM (BAC) (red).The results show firstly the strong signal that is obtaineddemonstrating the detection of HAV-specific dIgA, and secondly that thissignal is specific for dIgA not IgM because the IgM depletion method didnot substantially reduce the level of HAV-specific reactivity comparedto untouched serum, in contrast to the results shown in FIG. 12 for IgMdetection.

FIG. 14 provides the results of ELISA demonstrating the detection ofHAV-specific dIgA in R/HpIgR capture, using sera from patients with orwithout acute HAV infection (Accurun HAV panel, positive (POS), lowpositive (LOW POS), or negative (NEG)). The results show the strongdetection of HAV-specific dIgA in all POS samples and in one of two LOWPOS samples, with minimal background reactivity in NEG samples,demonstrating the utility of R/HpIgR capture of antigen-specific dIgAfor the diagnosis of acute HAV infection.

FIG. 15 provides the results of ELISAs demonstrating the detection ofhepatitis E virus (HEV)-specific dIgA in R/HpIgR capture or HpIgRcapture, using sera from patients with or without acute HEV infection.On the left, the ELISA OD of individual samples is shown, demonstratingthe utility of R/HpIgR capture of antigen-specific dIgA for thediagnosis of acute HEV infection, with lower but still significantutility of HpIgR capture for this purpose, and negligible backgroundreactivity in either example. On the right, the reactivity of serialdilutions of each serum sample is shown, confirming the utility ofR/HpIgR capture and lower utility of HpIgR capture for diagnosis ofacute HEV infection. It is likely that the lower utility of HpIgRcapture in these examples is due to the much higher overallconcentration of IgM in serum versus dIgA, resulting in only a lowproportion of the IgM captured by HpIgR being specific for HEV.

FIG. 16 is a schematic of a second preferred method for detection ofantigen-specific dIgA (or IgM), in which antigen is coated directly ontothe ELISA plate (in this case, hepatitis E virus (HEV) antigen). Serumsamples are applied to the plate and antigen-specific antibodies,including IgM and dIgA, bind to the antigens and are then detected witheither anti-IgM HRP, or R/HpIgR and anti-human SC HRP. After finalwashing, signal is generated with TMB substrate.

FIG. 17 provides the results of a comparison of HEV-specific dIgA versusHEV-specific IgM. Both methods are able to detect all HEV-infectedpatients with strong ELISA signals, compared to extremely low backgroundfor control (HEV-negative) patients in the dIgA assay, and lowbackground in the IgM assay. Notably, some samples show higher levels ofdIgA compared to IgM (sample J13, J7), while others show higher levelsof IgM compared to dIgA (J4, J11). This demonstrates that the dIgA andIgM responses in patients are independent, and suggests that acombination of both IgM and dIgA detection may be useful in somedesirable assay formats.

FIG. 18 provides the results of a comparison of HEV-specific dIgA versusHEV-specific IgM using sera that are either untouched, or substantiallydepleted of IgM using Capture-Select IgM, and then serially diluted. Theresults confirm that the IgM assay is specific for IgM, because thereactivity is ablated by IgM depletion, whereas the dIgA assay ispredominantly specific for dIgA and not IgM, because the reactivity isonly slightly affected by IgM depletion.

FIG. 19 provides the results of a comparison of HEV-specific dIgA versusHEV-specific IgM using sera that are either untouched, or substantiallydepleted of IgM using Capture-Select IgM. The results confirm that theIgM assay is specific for IgM, because the reactivity is ablated by IgMdepletion, whereas the dIgA assay is predominantly specific for dIgA andnot IgM, because the reactivity is only slightly affected by IgMdepletion.

FIG. 20 provides the results of comparison of HEV-specific dIgA versusHEV-specific IgM using sera that are either untouched, or substantiallydepleted of IgM using Capture-Select IgM. The results confirm that theIgM assay is specific for IgM, because the reactivity is ablated by IgMdepletion, whereas the dIgA assay is predominantly specific for dIgA andnot IgM, because the reactivity is only slightly affected by IgMdepletion. The reduction in dIgA activity following IgM depletion isstatistically significant when using a paired T-test to compare samplesbefore and after depletion, but is not significant when using aMann-Whitney test to compare the overall sample sets before and afterdepletion.

FIG. 21 provides the results of ELISA demonstrating that the R/HpIgR canbe used in both capture (A) and detection (B) of mouse dimeric IgA, withnegligible background reactivity to monomeric (human) IgA. A. Dilutionsof purified mouse IgA monoclonal antibody 3H1 (anti-HAV) or purifiedmonomeric human IgA were coated on plates and detected with R/HpIgR andanti-SC antibodies. B. R/HpIgR was coated on plates and dilutions ofpurified mouse IgA monoclonal antibody 3H1 or purified monomeric humanIgA were allowed to bind overnight, then detected with anti-mouse IgA oranti-human IgA. The binding of IgA from diverse species to human orrabbit pIgR is known in the art, and this demonstrates that the novelpIgR strategy described herein has utility for diagnosis of infection inother species. It is also useful for purification of dIgA from otherspecies—e.g., purification of mouse, rabbit or rate IgA monoclonalantibodies versus jacalin agarose.

FIG. 22 provides the results of ELISA demonstrating that the R/HpIgR isequally effective for detection of mouse dimeric IgA and human dimericIgA, with negligible background reactivity to monomeric human IgA. A.Dilutions of purified mouse IgA monoclonal antibody 3H1 (anti-HAV) orpurified dimeric or monomeric human IgA were coated on plates anddetected with R/HpIgR and anti-SC antibodies.

FIG. 23 provides a model outlining the pathogenic consequences of acutehuman immunodeficiency (HIV) infection, which leads to rapid CD4depletion in the gut as well as the periphery, with a subsequentreduction in gut barrier function, increased leakage of gut contents andmicrobial translocation, leading to increased immune activation whichdrives pathogenesis and further reduction of CD4 T-cell levels(Brenchley et al, Nat Med 12: 1365-1371, 2006). There is an unmet needfor simple, standardised assays that can detect one or more of thesesteps in the pathogenesis pathway so that appropriate interventions canbe provided to patients, with only CD4 testing having been integratedinto the standard of care for HIV-infected patients. Detection of CD4depletion in the gut requires endoscopy; detection of decreased gutbarrier function requires complicated sugar challenge studies or othermethods; detection of gut leakage and microbial translocation can beachieved using markers such as bacterial LPS or 16sRNA in serum butresults are highly variable due in part to the wide variation in gutmicrobiota between individuals; immune activation requires complex Flowcytometry protocols that are difficult to standardise acrossinstruments/operators.

FIG. 24 is a schematic of the increase in microbial translocation due togut leakage induced by pathogenic HIV or SIV infection, compared tonormal low levels of translocation in nonpathogenic SIV infection.

FIG. 25 illustrates one expected consequence of increased microbialtranslocation is the induction of increased IgA responses due to mucosalantigen exposure. French et al., J Infect Dis. 200(8): 1212-1215, 2009demonstrated that indeed the total level of IgA in HIV patients after 6years of follow up was inversely correlated with the level of CD4T-cells in patients undergoing highly active antiretroviral therapy,suggesting that even in patients being treated with the most effectivecurrent antiviral therapies, microbial translocation contributes topathogenesis. However these results also show that total IgA is highlyvariable between individuals, and does not provide a prognostic markerthat can be used in management of individual patients.

FIG. 26 provides a schematic illustration of the increase in microbialtranslocation due to gut leakage induced by pathogenic HIV or SWinfection, compared to normal low levels of translocation innonpathogenic SIV infection, showing the expected effect on dimeric IgAand secretory IgA levels in the plasma compartment. Under normalconditions or nonpathogenic SIV infection, gut barrier integrity ismaintained and the level of SIgA in the lumen of the gut reflects theamount of its precursor dIgA in the lamina propria. Only a minimalamount of SIgA is returned to the plasma compartment, either throughactive transport by M-cells in the gut, or a small amount of gutleakage. The amount of leakage or active transport can be estimated bycomparing the serum/plasma concentration of SIgA to that of itsprecursor dIgA, giving a ratio of SIgA/dIgA. Under conditions ofpathogenic HIV or SIV infection, or other physiological challenges thatresult in gut leakage, the total amount of dIgA is likely to be somewhatelevated and may lead to higher levels of SIgA secretion into the lumen.However a much higher proportion of SIgA will be returned to the plasmacompartment due to passive leakage through the compromised gut barrier,resulting in an elevated SIgA/dIgA ratio.

FIG. 27 provides a schematic of one of several typical assays that canbe used to measure the relative amount of different IgA forms in orderto estimate the SIgA/dIgA ratio. In this example, the amount of dIgA ismeasured by capture of dIgA using R/HpIgR, and detection usingmonoclonal antibodies against either IgA1, or IgA2, or against both IgAsubclasses. Monomeric IgA does not bind to pIgR; SIgA does bind toR/HpIgR but with lower affinity than dIgA and can be removed by washingwith 3.5 M urea if desired. SIgA is measured in the same way but usinganti-SC antibody capture instead of R/HpIgR. The SIgA/dIgA ratio is thencalculated as a simple ratio of the assay reactivities for SIgA anddIgA.

FIG. 28 provides the results of ELISA demonstrating the detection ofhighly elevated SIgA2/dIgA2 (S/d) ratios in a proportion of HIV-infectedpatients, compared to the majority of HIV-infected patients and allcontrol subjects (magenta). The assay cutoff for elevated SIgA/dIgA wasset as the mean plus 3 standard deviations of the SIgA/dIgA ratio amongnon-HIV control subjects, and 7/30 HIV-infected subjects showedSIgA/dIgA ratios above this cutoff. Notably, the range of SIgA/dIgAratios among normal subjects is smaller than the range for SIgA or dIgAalone, because the role of dIgA as the precursor of SIgA provides anormalising effect for each patient.

FIG. 29 provides the results of ELISA demonstrating the total amount ofSIgA in patient and control sera (arbitrary units). The amount of SIgA2in normal patients varies over an 11-fold range, but all normal controlsfall within a cutoff of the mean plus 3 standard deviations. The amountof SIgA2 in HIV-infected patients varies over a slightly larger range(16-fold), but only 2/30 patients are above the cutoff range. Among theHIV-infected patients, those patients who demonstrated elevatedSIgA2/dIgA2 ratios in FIG. 28 are indicated with red markers. It can beseen that these patients with elevated SIgA2/dIgA2 ratios are foundthroughout much of the normal range of the total SIgA2 signal, andcannot be distinguished from the normal controls on the basis of thetotal SIgA2 alone. This confirms the utility of using SIgA/dIgA ratiosbecause the role of dIgA as the precursor of SIgA provides a normalisingeffect for each patient. The R/HpIgR system provides the utility formeasuring this ratio.

FIG. 30 illustrates the correlation of SIgA2/dIgA2 ratio versus theimmune activation marker, CD8+ HLA-DR+ CD38+ T-cells, in a differentHIV-infected population to that shown in FIGS. 28 and 29. While theoverall correlation is low, it is apparent that patients withSIgA2/dIgA2 ratios of >4 in this experiment have elevated levels ofimmune activation markers (p<0.0001).

FIG. 31 illustrates the correlation of SIgA1/dIgA1 ratio versus theimmune activation marker, CD8+ HLA-DR+ CD38+ T-cells, in the samepopulation as FIG. 30. While the overall correlation is lower again thanfor IgA2, it is apparent that patients with SIgA1/dIgA1 ratios of >10 inthis experiment have elevated levels of immune activation markers(p<0.015). The lower correlation for IgA1 and higher cutoff ratio (10versus 4) for significance highlights the value of specificallymeasuring IgA2 because of its predominant site of synthesis in the gut,being the tissue in which leakage of SIgA is likely to be clinicallyrelevant marker of gut leakage and immune activation.

FIG. 32 illustrates the correlation of SIgA1/dIgA1 ratio versusSIgA2/dIgA2 ratio, in the same population as FIGS. 30 and 31. WhileSIgA1/dIgA1 ratios are significantly correlated with SIgA2/dIgA2, it isnotable that there are some patients with highly elevated SIgA1/dIgA1ratios and relatively low SIgA2/dIgA2 ratios. This suggests that theremay be some value in measuring IgA1, or total IgA, in addition to IgA2in the calculation of SIgA/dIgA ratios as a measure of gut leakage andimmune activation.

FIG. 33 provides the nucleotide sequence and amino acid sequence ofCHIMERA-CD4 cyto (R/HpIgR-cyto) showing rabbit sequences underlined andhuman sequences in black, and CD4 cyto sequences in grey.

FIG. 34 provides the nucleotide sequence and amino acid sequence ofCHIMERA (R/HpIgR) showing rabbit sequence underlined and human sequencein black.

FIG. 35 provides graphical and tabulated date showing hepatitis C virusspecific dIgA detection. In addition to the acute, self-limitinghepatitis A virus and hepatitis E virus which are transmitted fromperson to person via fecal-oral routes, there are at least three otherviruses that cause viral hepatitis which is commonly chronic and leadingto severe long-term disease, and where the viruses are considered to beblood-borne, namely hepatitis B, hepatitis C and hepatitis D. In thisexample, it can be seen that even for a chronic infection such ashepatitis C virus, HCV-specific dIgA is detectable for only a relativelyshort time after infection, up to around 100 days after the last bloodsample that tested negative for HCV, suggesting that HCV-specific dIgAmay be a marker of acute infection. While only 4 out of 5 (80%) of HCVpatients had detectable HCV-specific dIgA in this assay, it will beevident to those skilled in the art that there are a variety of methodsthat could be used to increase the sensitivity of this assay fordetection of acute-phase HCV, which will be useful in determining thebest options for treating patients with antiviral drugs orinterferon-containing medications. It will also be evident that sincevirus-specific dIgA is found in the diverse range of diseases includinghepatitis A, hepatitis E, hepatitis C and tuberculosis, it is reasonableto assume that it may be detectable, and of utility in diagnostic andtherapeutic protocols, for any other infectious disease.

FIG. 36 provides a graphical representation of data comparing differentcommercially available antibodies for binding to R/HpIgR or HpIgR. Itwill be apparent to those skilled in the art that there are manydifferent monoclonal and polyclonal antibodies directed against the pIgR(also known as secretory component or SC) that may be used for thedetection of bound pIgR, and thus antigen-specific or total dIgA or IgM,in a variety of assay methods. In this example, it can be seen thatseveral commercially available antibodies can be used for this purpose,whereby serial dilutions of either recombinant expressed chimericR/HpIgR or recombinant expressed human HpIgR was coated on polystyreneELISA plates, and detected using serial dilutions of mouse monoclonal orsheep polyclonal antibodies, and detected with appropriate anti-speciesantibodies. Note that the R/HpIgR is present at higher concentrations,and so there is no titration of the assay signal in this experiment,whereas with the HpIgR which is present at lower concentrations, thetitration of antibody reactivity reveals that commercial Abcammonoclonal antibody 17921 is approximately equivalent to sheeppolyclonal antibody, while Abcam 17377 and 3924 appear to be lessreactive, as does the Nordic Immunology monoclonal against SC. Therabbit antibody is a negative control. These and other antibodies can beselected according to their binding to either free pIgR forms, or pIgRforms already bound to dIgA or pIgM, to fit the purpose needed indetection of R/HpIgR or HpIgR or dIgA-binding variants thereofaccordingly.

DESCRIPTION OF PARTICULAR EMBODIMENTS

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or element or method step or group of integers orelements or method steps but not the exclusion of any other integer orelement or method step or group of integers or elements or method steps.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of”. Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they affect theactivity or action of the listed elements.

As used herein the singular forms “a”, “an” and “the” include pluralaspects unless the context clearly dictates otherwise.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A summary of sequence identifiers is provided in Table 1. Asequence listing is provided at the end of the specification.

As described herein, the present disclosure provides an antibody captureprocess comprising determining the level or presence of dimeric orpolymeric IgA (dIgA) in a biological sample. The process of the presentinvention may be practised by detecting only the level or presence ofdIgA, or it may be practised in combination with protocols to determinethe level or presence of one or more further antibody forms (e.g.,monomeric, dimeric, polymeric or pentameric complexes), classes(isotypes) or subclasses (e.g., dIgA1, dIgA2, SIgA2, etc.). In someembodiments, the process is practised using specific antigens that bindto dIgA molecules of interest. In other embodiments, the process may bepractised to identify antigens of interest which engender dIgA responsesin a subject that may be detected in a sample from a subject. In someembodiments, the process enables the development inter alia ofdiagnostic assays and therapeutic protocols that are useful forassessing secretory IgA responses at mucosal surfaces and associatedtissues such as gut-associated lymphoid tissue (GALT).

The present process is predicated in part on the ability to detect dIgAor dIgA and IgM with high sensitivity and specificity in binding assaysusing a recombinant polymeric Ig receptor (pIgR). The present processemploys recombinant pIgR or recombinant variants of pIgR that bind dIgAand IgM, as well as recombinant pIgR or variants of pIgR thatpreferentially bind dIgA and substantially fail to bind IgM.

Accordingly, the present specification provides an antibody-captureprocess comprising detecting or capturing a precursor to secretory dIgA(SIgA), namely dIgA. In some embodiments, the process comprises step (i)contacting a biological sample from a subject with recombinant polymericimmunoglobulin (Ig) receptor (pIgR) or an dIgA-binding variant thereof,wherein the pIgR or variant binds dIgA and IgM; or wherein the pIgR orvariant binds dIgA and substantially fails to bind IgM, and wherein thepIgR substantially does not bind monomeric IgA, and step (ii)determining the level or presence of dIgA that has bound to pIgR. Insome embodiments, step (ii) comprises detecting a complex between dIgAand pIgR or a complex between bound dIgA and an antigen.

Reference herein to “biological sample” includes a sample obtained froma subject comprising antibodies. The term also includes samplecomprising cells expressing dIgA e.g., hybridoma cells, and samplescomprising recombinant dIgA expressed from cell lines cultured in vitro.Biological samples from subjects include blood and serum samples, otherbodily liquids, biopsy etc. Blood and serum samples are preferred.Reference to an “antigen” includes a protein or infection agent or partof a protein or part of an infection agent, as known in the art.Reference to R/HpIgR includes chimeric forms comprising a immunoglobulindomain from a rabbit pIgA sequence or similar dIgA-binding variantsequences derived from rat or mouse or functional (dIgA-binding)variants thereof. Thus R includes rabbit, or mouse, or rat-derivedsequences.

Determining the presence or level of dIgA or pIgR or a complex betweendIgA and recombinant pIgR or a complex between recombinant dIgA and anantigen may be by any convenient protocol.

A diverse range of assays are used in research, analysis, developmentand clinically to detect analytes of interest. Immunoassays are aparticularly useful form of assay that exploits the specificity,strength and diversity of antibody-antigen type or protein-proteinreactions to analyse samples and detect specific components therein. Awide range of immunoassay techniques are available, such as thosedescribed in Wild D. “The Immunoassay Handbook” Nature Publishing Group,2001.

Methods of detecting antibody complexes, antigens or antibody-ligandcomplexes are well known in the art. For example, the enzyme-linkedimmunosorbent assay (ELISA) and radioimmunoassay (RIA) are routinelyused in laboratories. These methods generally require some level ofskill in laboratory techniques. A variety of methods have also beendeveloped which require little skill and are rapid to perform, and whichare therefore suitable for the detection of antibody to specificantigens at the point of care or analysis. In particular,immunochromatographic or dipstick enzyme-linked immunosorbent kits havebeen developed to assay for a number of infections agents.

Immunochromatographic devices are expressly contemplated comprisingdIgA-binding reagents such as recombinant pIgR and further comprisingantigens of interest identified as described herein as binding dIgA frominfected subjects or subjects exhibiting mucosal immune activation.

Kits or immunochromatographic devices comprise, for example,reverse-flow or lateral-flow formats.

In an illustrative embodiment, a kit for assessing immune status in abiological sample from a subject is provided which employs one or moreantigens of interest recognised by dIgA from subjects with activeinfections or conditions associated with mucosal immune activation, andemploys a pIgR molecule or dIgA-binding variant thereof as adIgA-binding reagent. In a preferred embodiment, the antigen is not a TBantigen.

In some embodiments, the kit comprises:

-   a) an immunographic device comprising a porous membrane operably    connected to a sample portion, a test portion, and optionally a    control portion; and further comprising a sucker portion, portion    comprising a pIgR molecule or dIgA-binding variant thereof, a    portion comprising an antigen or agent of interest and optionally a    conjugate portion; and-   b) instructions for using the immunographic device to detect the    presence of antigen specific dIgA antibody in the sample.

In one embodiment, the pIgR or dIgA-binding variant thereof is HpIgR orR/HpIgR or a dIgA binding variant thereof.

The subject assays may employ a wide range of suitable detection markersknown in the art. In some embodiments, the detection marker may bedetected using detectable characteristics of the detection marker and awide range of detection protocols using detectable markers are wellknown to those of ordinary skill in the art. In some embodiments, thedetection marker is directly or indirectly bound or otherwise associatedwith an antigen or infectious agent of interest. In other embodiments,the dIgA binding agent, such as pIgA comprises or is designed tointeract with a detection marker. In some embodiments, the detectionmarker is connected the antigen or dIgA binding agent using bindingpartners known in the art such as without limitation biotin:avidin oranti-biotin antibody:biotin.

Polymeric immunoglobulin receptor (pIgR) is encoded by the PIGR gene andis expressed in mucosal epithelial cells where it facilitates uptake ofdIgA and secretion of SIgA. pIgR has five immunoglobulin-like domainswhich bind to dIgA including to the J-chain thereof. pIgR also binds topentameric IgM.

As determined herein it is possible to detect both dIgA and IgM withhigh sensitivity and specificity using recombinant human polymeric Igreceptor and parts and variants thereof (see FIG. 3). In one particularnon-limiting embodiment it is shown herein that dIgA can be selectivelydetected using a recombinant form of the polymeric Ig receptor having atleast domain 1 derived from the rabbit pIgR, for example, a chimera ofrabbit (domain 1) and human (domain 2-5) pIgRs, or with all domains fromrabbit pIgR. In some embodiments, the recombinant pIgR described hereinare designed to bind preferentially to dIgA (plus or minus IgM), and canbe used either to capture dIgA (IgM) specifically to a solid phase forreaction with an antigen of interest, in which case the pIgR does notneed to have an associated detection reagent, or alternatively to detectthe presence of dIgA (±IgM) bound to an antigen of interest immobilizedon a solid phase, in which case the pIgR may be conveniently detectedusing antibodies or other reagents directed against the pIgR itself, oragainst epitope tags or other sites introduced into the recombinant pIgRusing methods well known in the art. A further advantage of pIgR is thatit shows very low background reactivity in assays, unlike typicalantibody-based detection reagents.

Roe et al., J Immunol 162: 6046-52, 1999 describe a chimeric pIgRcomprising immunoglobulin-like domain 1 (D1) derived from rabbit, andD2-5 derived from human pIgR which has preferential binding to dIgA overIgM. However, they do not disclose or suggest the use of this form orany other pIgR variant for detection or binding only of dIgA fordiagnostic purposes or the advantages of the recombinant pIgA or dIgAbinding variants disclosed herein. The substitution of human for rabbit(or mouse or rat) D1 provides preferential binding of dIgA, but it wouldbe expected that substitution of any one or more of D2-D5 may also besubstituted with the rabbit sequence to give a molecule thatpreferentially binds to dIgA and these variants are also encompassed.Accordingly, in some embodiments, any one or more of D1, D2, D3, D4 orD5 is substituted with rabbit, mouse or rat homologs.

In some embodiments, the recombinant pIgR lacks a transmembrane domain(ATM). In other embodiments, the recombinant pIgR lacks a cytoplasmicdomain. In some embodiments, the recombinant pIgR lacks a TM domain anda cytoplasmic domain (ACYT). In some embodiments, recombinant pIgRcomprises a substitution in the cytoplasmic domain and provides a CD4cytoplasmic domain. Various forms of recombinant pIgR are contemplatedand illustrative examples are illustrated in FIGS. 4 to 7, furtherdescribed in the figure legends. The ability to design and testrecombinant pIgR having a desired level of specific dIgA is illustratedin FIG. 8 and described in the legend to FIG. 8.

Phillips-Quagliata et al., J Immunol 165: 2544-2555, 2000 indicate thatboth rat and mouse bind predominantly dIgA, but that for mouse there isa form expressed on B-cells that has only a single amino acid change butbinds both IgM and dIgA—to quote from page 2552:

“Although human pIgR and the T560 mouse pIgR bind both pIgA and IgM,rabbit (44) and rat (47) hepatocyte pIgR bind only pIgA well and do nottranslocate IgM into bile. Because mouse liver similarly translocatespIgA but not IgM into bile (48, 49), it is generally assumed that mousehepatocyte pIgR resembles rat and rabbit pIgR and binds IgM poorly ornot at all. If this is true, then the difference between the mousehepatocyte and the T560 pIgR that makes the latter behave more likehuman pIgR must be explained. Given that the amino acid sequences of themouse hepatocyte and T560 B cell pIgRs are the same except for the Valto Ala change in domain 2, the difference most likely reflectsdifferential folding or glycosylation of the pIgR, probably the latter.It is easy to imagine that a bulky carbohydrate on hepatocyte-derivedpIgR could interfere with IgM but not with IgA binding. Furthermore, ithas already been shown that deglycosylation of human SC allows it toinhibit binding of biotinylated native SC to pIgA with 10 times greaterefficiency than native SC itself (50), suggesting that some of thecarbohydrate moieties on human pIgR may actually impede binding even ofpIgA.”

Accordingly, in some embodiments, a deglycosylated variant of therecombinant pIgR including R/HpIgR is used to improve binding affinityto dIgA. In some embodiments, this may be achieved by expressing thepIgR in a glycan-deficient cell line known in the art such as, forexample, a glycan deficient CHO cell line.

In plural embodiments, recombinant pIgR comprises a deletion in thetransmembrane domain (ATM) to allow for convenient secretion of therecombinant protein and ease of use as a diagnostic/prognostic/screeningagent.

In some embodiments, the recombinant pIgR comprises a heterologousdetection domain.

Multiple detection domains are known in the art and are encompassed.

In some embodiments, the recombinant pIgR comprises a heterologousbinding domain.

Multiple binding domains are known in the art and are encompassed.

In other embodiments, the recombinant pIgR is bound to a solid support.Solid supports include plates, wells, beads, agarose particles,nitrocellulose strips, etc.

In some embodiments, recombinant pIgR is produced in glycan deficientcells such as glycan deficient CHO cells to enhance preferential bindingto dIgA over IgM.

In some embodiments, the recombinant pIgR is derived from a primate suchas human pIgR and comprises at least one immunoglobulin-like domainderived from a non-primate such as rabbit, mouse, rat.

In some embodiments, the recombinant pIgR comprises an amino acidsequence set out in SEQ ID NO:2, or SEQ ID NO: 4, or SEQ ID NO: 6, orSEQ ID NO: 12, or SEQ ID NO: 14, or SEQ ID NO: 16, or an dIgA-bindingpart thereof or and a dIgA binding variant thereof. Illustrativevariants comprise at least 70% amino acid sequence identity to one ofSEQ ID NO: 2, 4, 6, 12, 14 or 16 or deletion variants thereof lacking acytoplasmic domain.

Variants include deletion, substitution and insertional variants.Illustrated herein are human derived pIgR varied by one or moreimmunoglobulin domains (D). Variants include “parts” which includesfragments comprising from about 50%, 60%, 70%, 80%, 85%, 90%, 95% of thereference sequence. Substitution for an equivalent domain from a lowermammal such as a rat, mouse or rabbit domain.

“Variants” of the recited amino acid sequences are also contemplated.Variant molecules are designed to retain the dIgA binding functionalactivity of the pre-modified recombinant pIgR or to exhibit enhancedactivity. Polypeptide variants according to the invention can beidentified either rationally, or via established methods of mutagenesis(see, for example, Watson, J. D. et al., “Molecular Biology of theGene”, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987).Random mutagenesis approaches require no a priori information about thesequence that is to be mutated. This approach has the advantage that itassesses the desirability of a particular mutant based on its function,and thus does not require an understanding of how or why the resultantmutant protein has adopted a particular conformation. Indeed, the randommutation of target gene sequences has been one approach used to obtainmutant proteins having desired characteristics (Leatherbarrow R., J.Prot. Eng., 1:7-16, 1986; Knowles J. R., Science, 236:1252-1258, 1987;Shaw W. V., Biochem. J., 246:1-17, 1987; Gerit J. A., Chem. Rev.,87:1079-1105, 1987). Alternatively, where a particular sequencealteration is desired, methods of site-directed mutagenesis can beemployed. Thus, such methods may be used to selectively alter only thoseamino acids of the protein that are believed to be important (Craik C.S., Science, 228:291-297, 1985; Cronin et al., Biochem., 27: 4572-4579,1988; Wilks et al., Science, 242:1541-1544, 1988). Illustrative aminoacids affect glycosylation of the recombinant pIgR. Polypeptides,resulting from rational or established methods of mutagenesis or fromcombinatorial chemistries, may comprise conservative amino acidsubstitutions. It is well understood in the art that some amino acidsmay be changed to others with broadly similar properties withoutchanging the nature of the activity of the polypeptide (conservativesubstitutions, see Table 3).

Variant pIgR polypeptides comprises at least 50% sequence identity toherein amino acid sequence at least over the immunoglobulin-like domainregion.

The terms and “sequence identity” as used herein refer to the extentthat sequences are identical or functionally or structurally similar onan amino acid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity”, for example, is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical amino acidresidue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp,Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequencesto yield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparison(i.e., the window size), and multiplying the result by 100 to yield thepercentage of sequence identity. For the purposes of the presentinvention, “sequence identity” will be understood to mean the “matchpercentage” calculated by the DNASIS computer program (Version 2.5 forwindows; available from Hitachi Software engineering Co., Ltd., SouthSan Francisco, Calif., USA) using standard defaults as used in thereference manual accompanying the software. Similar comments apply inrelation to sequence similarity which counts as identical, substitutionsinvolving conservative substitutions.

Preferably, the percentage similarity between a particular sequence anda reference sequence (nucleotide or amino acid) is at least about 60% orat least about 70% or at least about 80% or at least about 90% or atleast about 95% or above such as at least about 96%, 97%, 98%, 99% orgreater. Percentage similarities or identities between 60% and 100% arealso contemplated such as 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

In another embodiment there is provided recombinant pIgR encoded by thesequence of nucleotides set out in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15, or a dIgA-binding andoptionally IgM-non binding variant thereof having at least 60%nucleotide sequence identity thereto or at least 60% nucleotide sequenceidentity to deletion variants thereof lacking a cytoplasmic domain.

In some convenient embodiments, the recombinant pIgR is a humanrecombinant pIgR variant comprising at least one immunoglobulin-likedomain derived from a rabbit.

In some embodiments, (human) pIgR that binds pIgM and dIgA is employed.In some embodiments, IgG and/or IgM, are depleted using known protocols,and, if IgM is depleted then the human pIgR is selective for dIgA amongwhat is left in the sample. In some embodiments, for diagnosis of manyinfections it may be preferable to detect both dIgA and pIgM—certainlyIgA plus IgM is reported in the literature for hepatitis E and asillustrated herein there are some samples for hepatitis A or hepatitis Ein which either IgM or dIgA are much stronger, suggesting that theircombined detection is useful. While this could be achieved using amixture, for example, of anti-IgM and the R/HpIgR, it may moreconveniently be achieved with a mixture of HpIgR and R/HpIgR (so thatthe IgM does not outcompete the dIgA), or with HpIgR alone if it ispresent in excess over the sum of IgM and dIgA. It is proposed to bepreferable to use two variations of the same reagent rather than oneantibody plus a recombinant protein. In particular, pIgR is morethermally stable than antibody which is an advantage for testproduction.

The use of recombinant pIgR is also highly advantageous not leastbecause the reagent displays low background (at least 50% lessbackground compared to antibody based reagents) in binding assays unlikemost antibody based binding agents. In addition, recombinant pIgRdisplays high thermal stability. For example, lyophilised recombinantpIgR retained 50% activity at 60° C. and 100% activity at 45° C. afterthree weeks prior to reconstitution, which compares favourably to therapid loss of activity for dried anti-IgM antibody under the sameconditions.

In embodiments, where substantially only dIgA (or IgM) is to bedetected, recombinant human pIgR or dIgA and IgM binding variants may beemployed as the binding reagent, but specifically detection of bounddIgA (either dIgA1 or dIgA2) is achieved using anti-IgA1/IgA2, in thisembodiment, the presence of IgM is not a problem.

In some embodiments the process is for use in a method of assessingconditions or infections of a mucosal surface or associated tissues, orimmunity thereto. Illustrative applications to specific antigens aredescribed in the Figures and Figure legends for FIGS. 10 to 22 inclusiveand these general protocols are expressly contemplated herein as well asroutine variations thereto.

Illustrative mucosal surfaces include the upper and lower respiratorytracts, the gut and gut-associated lymphoid tissue, the genital tractand the liver.

Illustrative infections include without limitation those mediated bybacteria, viruses, parasites and other infectious organisms. In certainembodiments, infectious agents include, HIV, leprosy, syphilis,hepatitis (e.g., HEV, HAV, HCV) dengue virus, measles, rubella etc.

Illustrative conditions include diseases of organs such as therespiratory tract, lungs, gut, genital tract and liver. Gut conditionsinclude ulcerative colitis, Crohn's disease, IBS, leaky gut syndromeetc.

Reference to “subject” includes humans and a wide range of mammalian orother animals including wild and domesticated animals, pets, pests andpotential vehicles for emerging infectious diseases. In relation tosubjects, these may have an infection, they may have had exposure toinfection or they have had exposure to an infectious agent.

In one embodiment, FIG. 10 is a schematic of one preferred experimentalapproach for detecting the presence of antigen-specific dIgA in a samplesuch as human serum or plasma. Recombinant R/HpIgR-cyto is immobilisedon the ELISA plate, and incubated with serum or other samples. DimericIgA is captured on the solid phase, and after washing to remove othersample components (such as IgA and IgG that are not captured, left), thepresence of antigen-specific dIgA is detected by sequential addition ofantigen that is, for example, either biotinylated, or reacted with abiotinylated monoclonal antibody against the antigen, andstreptavidin-HRP. In this way, any antigen that is immobilised byreaction with antigen-specific dIgA will give a signal through thebiotin-streptavidin interaction or an equivalent reagent.

In one embodiment, FIG. 11 is a schematic of one preferred experimentalapproach for detecting the presence of hepatitis A virus-specific dIgAin serum (right), compared to detection of HAV-specific IgM using thestandard method of anti-IgM capture (left).

In one embodiment, FIG. 16 is a schematic of a second preferred methodfor detection of antigen-specific dIgA (or IgM), in which antigen iscoated directly onto the ELISA plate (in this case, hepatitis E virus(HEV) antigen). Serum samples are applied to the plate andantigen-specific antibodies, including IgM and dIgA, bind to theantigens and are then detected with either anti-IgM HRP, or R/HpIgR andanti-human SC HRP. After final washing, signal is generated with TMBsubstrate or equivalent reagent.

In another embodiment, FIG. 23 provides a model outlining the pathogenicconsequences of acute human immunodeficiency (HIV) infection, whichleads to rapid CD4 depletion in the gut as well as the periphery, with asubsequent reduction in gut barrier function, increased leakage of gutcontents and microbial translocation, leading to increased immuneactivation which drives pathogenesis and further reduction of CD4 T-celllevels (Brenchley et al, Nat Med 12: 1365-1371, 2006). There is an unmetneed for simple, standardised assays that can detect one or more ofthese steps in the pathogenesis pathway so that appropriateinterventions can be provided to patients, with only CD4 testing havingbeen integrated into the standard of care for HIV-infected patients.Detection of CD4 depletion in the gut requires endoscopy; detection ofdecreased gut barrier function requires complicated sugar challengestudies or other methods; detection of gut leakage and microbialtranslocation can be achieved using markers such as bacterial LPS or16sRNA in serum but results are highly variable due in part to the widevariation in gut microbiota between individuals; immune activationrequires complex Flow cytometry protocols that are difficult tostandardise across instruments/operators.

FIG. 24 provides a schematic illustration of the increase in microbialtranslocation due to gut leakage induced by pathogenic HIV or SWinfection, compared to normal low levels of translocation innonpathogenic SIV infection.

FIG. 25 illustrates that one expected consequence of increased microbialtranslocation is the induction of increased IgA responses due to mucosalantigen exposure. M. French et al Journal of Infectious Diseases 200;2009 demonstrated that indeed the total level of IgA in HIV patientsafter 6 years of follow up was inversely correlated with the level ofCD4 T-cells in patients undergoing highly active antiretroviral therapy,suggesting that even in patients being treated with the most effectivecurrent antiviral therapies, microbial translocation contributes topathogenesis. However these results also show that total IgA is highlyvariable between individuals, and does not provide a prognostic markerthat can be used in management of individual patients.

FIG. 26 provides a schematic of the increase in microbial translocationdue to gut leakage induced by pathogenic HIV or SIV infection, comparedto normal low levels of translocation in nonpathogenic SIV infection,showing the expected effect on dimeric IgA and secretory IgA levels inthe plasma compartment. Under normal conditions or nonpathogenic SIVinfection, gut barrier integrity is maintained and the level of SIgA inthe lumen of the gut reflects the amount of its precursor dIgA in thelamina propria. Only a minimal amount of SIgA is returned to the plasmacompartment, either through active transport by M-cells in the gut, or asmall amount of gut leakage. The amount of leakage or active transportcan be estimated by comparing the serum/plasma concentration of SIgA tothat of its precursor dIgA, giving a ratio of SIgA/dIgA. Underconditions of pathogenic HIV or SIV infection, or other physiologicalchallenges that result in gut leakage, the total amount of dIgA islikely to be somewhat elevated and may lead to higher levels of SIgAsecretion into the lumen. However a much higher proportion of SIgA willbe returned to the plasma compartment due to passive leakage through thecompromised gut barrier, resulting in an elevated SIgA/dIgA ratio.

In another embodiment, the present specification provides a process forassessing gut wall integrity. In some embodiments, and as discussedherein, this assessment provides a prognostic marker for HIV infectedpatients. In some embodiments, the process comprises determining therelative levels or a ratio of SIgA and dIgA in a sample from a humansubject. In some embodiments, the process comprises the step of (i)contacting a biological sample with recombinant polymeric immunoglobulin(Ig) receptor (pIgR) or an dIgA-binding variant thereof, wherein thepIgR or variant binds dIgA and IgM; or wherein the pIgR or variant bindsdIgA and substantially fails to bind IgM, and wherein the pIgRsubstantially does not bind monomeric IgA or secretory IgA and step (ii)determining the level or presence of dIgA that has bound to pIgR. Insome embodiments, step (ii) comprises detecting a complex between dIgAand pIgR or a complex between bound dIgA and an antigen essentially asdescribed herein. In another expression of this embodiment, thespecification contemplates a process of determining the dIgA/SIgA ratiofor use in assessing HIV infected subjects.

Secretory IgA (SIgA) is normally found in only trace amounts in plasma,but in patients with compromised gut barrier integrity, it is proposedherein that a larger proportion of SIgA will leak across the gut barrierand enter the plasma. The normal concentration of SIgA will differbetween individual patients, because patients have widely varying levelsof the SIgA precursor, dIgA. It is therefore useful to simultaneouslymeasure the concentration of both dIgA and SIgA, with the ratio of SIgAto dIgA in the individual patient providing a measure of gutintegrity/leakage.

FIG. 27 provides a schematic of one of several typical assays that canbe used to measure the relative amount of different IgA forms in orderto estimate the SIgA/dIgA ratio. In this example, the amount of dIgA ismeasured by capture of dIgA using R/HpIgR, and detection usingmonoclonal antibodies against either IgA1, or IgA2, or against both IgAsubclasses. Monomeric IgA does not bind to pIgR; SIgA does bind toR/HpIgR but with lower affinity than dIgA and can be removed by washingwith 3.5 M urea if desired. SIgA is measured in the same way but usinganti-SC antibody capture instead of R/HpIgR. The SIgA/dIgA ratio is thencalculated as a simple ratio of the assay reactivities for SIgA anddIgA.

As shown in the Examples, a proportion of patients infected with HIVexhibit an elevated level of SIgA compared to dIgA in patient plasma,consistent with elevated immune activation markers in these patients. Infurther embodiments, gut leakage can be determined by examining theindividual IgA isotypes, because IgA2 (dIgA2, SIgA2) represents around50% of IgA produced in the gut mucosa, but only around 10% of IgA inother tissues, and thus the ratio of SIgA2 to dIgA2 is likely to providea very sensitive measurement of gut leakage in an individual patient.

Accordingly, in some embodiments, the process comprises measuring thelevels of SIgA2 and dIgA2 and determining the ratio of SIgA2 to dIgA2wherein the ratio relative to a control is indicative of the presence orabsence or degree of gut leakage. In some embodiments, this ratioprovides marker for HIV infected patients thereby potentiallyfacilitating improved management of HIV infection in a subject. Theseassays are simple and have potential for high throughput as potentialfor incorporation into quantitative point of care devices.

Because the reaction between pIgR and dIgA is highly conserved acrossnot only mammals but all vertebrate species, it is proposed that thisprocess will have utility for detection of dIgA, and IgM when desirable,in a wide variety of species, providing a convenient and universalreagent for detection of immune activation or dIgA and/or IgM responsesin species such as bats and other wild or domesticated animals for whichthere may be no available anti-immunoglobulin reagents. This will beuseful in diagnosis of diseases of agricultural interest in domesticatedanimals, and for the diagnosis of disease and detection of hostreservoir species for emerging infectious diseases.

The present invention enables the use of the specific interactionbetween the recombinant expressed forms of pIgR and dIgA (plus or minusIgM), allowing pIgR to be used for specific binding or detection of dIgA(plus or minus IgM) to solid surfaces or other assay components asdesired.

In an embodiment, the herein described process and/or recombinant pIgRor variants thereof as described herein are sub-licensed for use inantigen screening or antibody selection and purification.

Treatment protocols are contemplated based upon the results of diagnosisor prognosis testing as described herein.

In some embodiments, the process further comprises: (a) generating datausing a process as described herein; (b) transforming the data intocomputer-readable form; and (c) operating a computer to execute analgorithm, wherein the algorithm determines closeness-of-fit between thecomputer-readable data and data indicating a diagnosis of a disease orcondition. In some embodiments, the algorithm comprises an artificialintelligence program, such as a fuzzy logic, cluster analysis or neuralnetwork. The subject methods may also be used in a personalized or apopulation medicine approach in the management of pathology platforms.

The present disclosure provides a computer program and hardware fordiagnosis in a subject once off, over time or in response to treatmentor other affectors. Values are assigned to complex levels which arestored in a machine readable storage medium. A computer program productis one able to convert such values to code and store the code in acomputer readable medium and optionally capable of assessing therelationship between the stored data and incoming data and optionally aknowledge database to assess a potential TB status and/or pneumonia.

The present specification therefore provides a web-based system wheredata on levels of complex are provided by a client server to a centralprocessor which analyses and compares to a control and optionallyconsiders other information such as patient age, sex, weight and othermedical conditions and then provides a diagnostic report.

The assay may, therefore, be in the form of a kit or computer-basedsystem which comprises the reagents necessary to form and detect theherein described antibody complexes and the computer hardware and/orsoftware including an algorithm to facilitate determination andtransmission of reports to a clinician.

The present invention contemplates a method of allowing a user todetermine the status of a subject with respect to TB, the methodincluding:

-   (a) receiving data from the conduct of the process as herein    described from the user via a communications network;-   (b) processing the subject data via multivariate analysis to provide    a diagnostic index value;-   (c) determining the status of the subject in accordance with the    index value in comparison with predetermined values; and-   (d) transferring an indication of the status of the subject to the    user via the communications network.

Conveniently, the method generally further includes:

-   (a) having the user determine the data using a remote end station;    and-   (b) transferring the data from the end station to the base station    via the communications network.

As used herein, the term “binds specifically,” and the like whenreferring to an antigen-binding molecule refers to a binding reactionwhich is determinative of the presence of an antigen in the presence ofa heterogeneous population of proteins and other biologics. Thus, underdesignated immunoassay conditions, the specified antigen-bindingmolecules bind to a particular antigen and do not bind in a significantamount to other proteins or antigens present in the sample. Specificbinding to an antigen under such conditions may require anantigen-binding molecule that is selected for its specificity for aparticular antigen. For example, antigen-binding molecules can be raisedto a selected protein antigen, which bind to that antigen but not toother proteins present in a sample. A variety of immunoassay formats maybe used to select antigen-binding molecules specificallyimmuno-interactive with a particular protein. For example, solid-phaseELISA immunoassays are routinely used to select monoclonal antibodiesspecifically immuno-interactive with a protein. See Harlow and Lane(1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,New York, for a description of immunoassay formats and conditions thatcan be used to determine specific immunoreactivity.

For example, specific recognition is provided by a primary antibody(polyclonal or monoclonal) and a secondary detection system is used todetect presence (or binding) of the primary antibody. Detectable labelscan be conjugated to the secondary antibody, such as a fluorescentlabel, a radiolabel, or an enzyme (e.g., alkaline phosphatase,horseradish peroxidase) which produces a quantifiable, e.g., colored,product. In another suitable method, the primary antibody itself can bedetectably labeled. For example, a protein-specific monoclonal antibody,can be used both as an immunoadsorbent and as an enzyme-labeled probe todetect and quantify complexes formed in the present process or kit.

The amount of such protein present in a sample can be calculated byreference to the amount present in a standard or reference preparationusing a linear regression computer algorithm (see Lacobilli et al.,(1988) Breast Cancer Research and Treatment 11:19-30). In otherembodiments, two different monoclonal antibodies to the protein ofinterest can be employed, one as the immunoadsorbent and the other as anenzyme-labeled probe.

Assays illustrated in the Examples are done in ELISA format with asingle antigen per well, per single antibody form or class or isotype.However there are well known methods where they could be combined into asingle assay for example using Luminex beads or similar where multipleindividual antigens are coated on beads having different intensity offluorescent label that can be discriminated in an instrument, and theamount of antibody binding to antigen on each bead can be separatelymeasured from the single sample. Similarly the Luminex beads can becoated with antibody or other reagents to capture the individualantibody forms or isotypes from a sample, and then labelled antigen (orantigens) is added and the different isotype reactivities are assessed.The same can be done in micro-arrays or other arrays. Having establisheduseful parameters in ELISA, it is then routine to transfer thesefindings to multiplex formats. In lateral flow and other point of caredevices, where the sample flows across a membrane, it is easy to havethe separate antigens present on the membrane as separate stripes orspots, and then detect the antibodies of one or more isotypes together;or else have different capture antibodies for the antibody isotopes, andthen detect the (labelled) antigen binding to each of the immobilizedantibody stripes or spots. The latter method (isotype capture, detectionof labelled antigen bound by the immobilised patient antibody) is a mostpreferred approach.

Additionally, recent developments in the field of protein capture arrayspermit the simultaneous detection and/or quantification of a largenumber of proteins. For example, low-density protein arrays on filtermembranes, such as the universal protein array system (Ge (2000) NucleicAcids Res. 28(2):e3) allow imaging of arrayed antigens using standardELISA techniques and a scanning charge-coupled device (CCD) detector.Immuno-sensor arrays have also been developed that enable thesimultaneous detection of clinical analytes. It is now possible usingprotein arrays, to profile protein expression in bodily fluids, such asin sera of healthy or diseased subjects, as well as in subjects pre- andpost-drug treatment.

Protein capture arrays typically comprise a plurality of protein-captureagents each of which defines a spatially distinct feature of the array.The protein-capture agent can be any molecule or complex of moleculeswhich has the ability to bind a protein and immobilize it to the site ofthe protein-capture agent on the array. The protein-capture agent may bea protein whose natural function in a cell is to specifically bindanother protein, such as an antibody or a receptor. Alternatively, theprotein-capture agent may instead be a partially or wholly synthetic orrecombinant protein which specifically binds a protein. Alternatively,the protein-capture agent may be a protein which has been selected invitro from a mutagenized, randomized, or completely random and syntheticlibrary by its binding affinity to a specific protein or peptide target.The selection method used may optionally have been a display method suchas ribosome display or phage display, as known in the art.Alternatively, the protein-capture agent obtained via in vitro selectionmay be a DNA or RNA aptamer which specifically binds a protein target(see, e.g., Potyrailo et al., (1998) Anal. Chem. 70:3419-3425; Cohen etal. (1998) Proc. Natl. Acad. Sci. USA 95:14272-14277; Fukuda, et al.(1997) Nucleic Acids Symp. Ser. 37:237-238; available from SomaLogic).For example, aptamers are selected from libraries of oligonucleotides bythe Selex™ process and their interaction with protein can be enhanced bycovalent attachment, through incorporation of brominated deoxyuridineand UV-activated crosslinking (photoaptamers). Aptamers have theadvantages of ease of production by automated oligonucleotide synthesisand the stability and robustness of DNA; universal fluorescent proteinstains can be used to detect binding. Alternatively, the in vitroselected protein-capture agent may be a polypeptide (e.g., an antigen)(see, e.g., Roberts and Szostak (1997) Proc. Natl. Acad. Sci. USA94:12297-12302).

An alternative to an array of capture molecules is one made through‘molecular imprinting’ technology, in which peptides (e.g., from theC-terminal regions of proteins) are used as templates to generatestructurally complementary, sequence-specific cavities in apolymerisable matrix; the cavities can then specifically capture(denatured) proteins which have the appropriate primary amino acidsequence (e.g., available from ProteinPrint™ and Aspira Biosystems).

Exemplary protein capture arrays include arrays comprising spatiallyaddressed TB antigens or antibody binding agents, which can facilitateextensive parallel analysis of numerous antigens and antibodies. Sucharrays have been shown to have the required properties of specificityand acceptable background, and some are available commercially (e.g., BDBiosciences, Clontech, BioRad and Sigma). Various methods for thepreparation of arrays have been reported (see, e.g., Lopez et al. (2003)J. Chromatogr. B 787:19-27; Cahill (2000) Trends in Biotechnology7:47-51; U.S. Pat. App. Pub. 2002/0055186; U.S. Pat. App. Pub.2003/0003599; PCT publication WO 03/062444; PCT publication WO03/077851; PCT publication WO 02/59601; PCT publication WO 02/39120; PCTpublication WO 01/79849; PCT publication WO 99/39210).

Immunoglobulin antigen-binding molecules are made either by conventionalimmunization (e.g., polyclonal sera and hybridomas), or as recombinantfragments, usually expressed in E. coli, after selection from phagedisplay or ribosome display libraries (e.g., available from CambridgeAntibody Technology, Biolnvent, Affitech and Biosite). Alternatively,‘combibodies’ comprising non-covalent associations of VH and VL domains,can be produced in a matrix format created from combinations ofdiabody-producing bacterial clones (e.g., available from Domantis).Exemplary antigen-binding molecules for use as protein-capture agentsinclude monoclonal antibodies, polyclonal antibodies, Fv, Fab, Fab′ andF(ab′)₂ immunoglobulin fragments, synthetic stabilized Fv fragments,e.g., single chain Fv fragments (scFv), disulfide stabilized Fvfragments (dsFv), single variable region domains (dAbs) minibodies,combibodies and multivalent antibodies such as diabodies and multi-scFv,single domains from camelids or engineered human equivalents.

Individual spatially distinct protein-capture agents are typicallyattached to a support surface, which is generally planar or contoured.Common physical supports include glass slides, silicon, microwells,nitrocellulose or PVDF membranes, and magnetic and other microbeads.

While microdrops of protein delivered onto planar surfaces are widelyused, related alternative architectures include CD centrifugationdevices based on developments in microfluidics (e.g., available fromGyros) and specialized chip designs, such as engineered microchannels ina plate (e.g., The Living Chip™, available from Biotrove) and tiny 3Dposts on a silicon surface (e.g., available from Zyomyx).

Particles in suspension can also be used as the basis of arrays,providing they are coded for identification; systems include colorcoding for microbeads (e.g., available from Luminex, Bio-Rad andNanomics Biosystems) and semiconductor nanocrystals (e.g., QDots™,available from Quantum Dots), and barcoding for beads (UltraPlex™,available from Smartbeads) and multimetal microrods (Nanobarcodes™particles, available from Surromed). Beads can also be assembled intoplanar arrays on semiconductor chips (e.g., available from LEAPStechnology and BioArray Solutions). Where particles are used, individualprotein-capture agents are typically attached to an individual particleto provide the spatial definition or separation of the array. Theparticles may then be assayed separately, but in parallel, in acompartmentalized way, for example in the wells of a microtiter plate orin separate test tubes.

In operation, a protein sample (see, e.g., U.S. Pat. App. Pub.2002/0055186), is delivered to a protein-capture array under conditionssuitable for protein or peptide binding, and the array is washed toremove unbound or non-specifically bound components of the sample fromthe array. Next, the presence or amount of protein or peptide bound toeach feature of the array is detected using a suitable detection system.The amount of protein bound to a feature of the array may be determinedrelative to the amount of a second protein bound to a second feature ofthe array. In certain embodiments, the amount of the second orsubsequent protein in the sample is already known or known to beinvariant.

In an illustrative example, fluorescence labeling can be used fordetecting protein bound to the array. The same instrumentation as usedfor reading DNA microarrays is applicable to protein-capture arrays. Fordifferential display, capture arrays (e.g. antibody arrays) can beprobed with fluorescently labeled proteins from or are labeled withdifferent fluorophores (e.g., Cy-3 and Cy-5) and mixed, such that thecolor acts as a readout for changes in target abundance. Fluorescentreadout sensitivity can be amplified 10-100 fold by tyramide signalamplification (TSA) (e.g., available from PerkinElmer Lifesciences).Planar waveguide technology (e.g., available from Zeptosens) enablesultrasensitive fluorescence detection, with the additional advantage ofno washing procedures. High sensitivity can also be achieved withsuspension beads and particles, using phycoerythrin as label (e.g.,available from Luminex) or the properties of semiconductor nanocrystals(e.g., available from Quantum Dot). Fluorescence resonance energytransfer has been adapted to detect binding of unlabelled ligands, whichmay be useful on arrays (e.g., available from Affibody). Severalalternative readouts have been developed, including adaptations ofsurface plasmon resonance (e.g., available from HTS Biosystems andIntrinsic Bioprobes), rolling circle DNA amplification (e.g., availablefrom Molecular Staging), mass spectrometry (e.g., available from SenseProteomic, Ciphergen, Intrinsic and Bioprobes), resonance lightscattering (e.g., available from Genicon Sciences) and atomic forcemicroscopy (e.g., available from BioForce Laboratories). A microfluidicssystem for automated sample incubation with arrays on glass slides andwashing has been co-developed by NextGen and Perkin Elmer Life Sciences.

In certain embodiments, the techniques used for detection of dIgA orother pre-selected products will include internal or external standardsto permit quantitative or semi-quantitative determination of thoseproducts, to thereby enable a valid comparison of the level orfunctional activity of these expression products in a biological samplewith the corresponding expression products in a reference sample orsamples. Such standards can be determined by the skilled practitionerusing standard protocols. In specific examples, absolute values for thelevel or functional activity of individual expression products aredetermined. Controls may include—individual and population control andsamples from diagnostic tests—an earlier time point.

In specific embodiments, the diagnostic method is implemented using asystem as disclosed, for example, in International Publication No. WO02/090579 and in copending PCT Application No. PCT/AU03/01517 filed Nov.14, 2003, comprising at least one end station coupled to a base station.The base station is typically coupled to one or more databasescomprising predetermined data from a number of individuals representingthe level TB antigen specific antibodies and their isotype structure(dimeric/polymeric) or subclass, when the predetermined data wascollected. In operation, the base station is adapted to receive from theend station, typically via a communications network, subject datarepresenting a measured or normalized level of at least one antibodytype in a biological sample obtained from a test subject and to comparethe subject data to the predetermined data stored in the database(s).Comparing the subject and predetermined data allows the base station todetermine the status of the subject in accordance with the results ofthe comparison. Thus, the base station attempts to identify individualshaving similar parameter values to the test subject and once the statushas been determined on the basis of that identification, the basestation provides an indication of the diagnosis to the end station. Inan embodiment, recombinant pIgR is sub-licensed for use in TB antigenscreening or TB serological diagnosis.

Each embodiment in this specification is to be applied mutatis mutandisto every other embodiment unless expressly stated otherwise.

Functionally equivalent methods and kits employing such methods areclearly within the scope of the invention as described herein.

The present invention is further described by the following non-limitingExamples.

EXAMPLE 1 ELISA Shows Preferential Binding of Chimeric pIgR to dIgA OverIgM, but Strong Binding of Human pIgR to IgM

As shown in FIG. 8, an ELISA is performed comparing the binding of HpIgRand R/HpIgR to human IgM and dIgA indicating preferential binding ofChimeric pIgR to dIgA over IgM, but strong binding of human pIgR to IgM.HpIgR or R/HpIgR were immobilised on 96-well Nunc Immulon platesovernight at 4° C. Dilutions of purified human IgM or dIgA in PBS werebound to the immobilised pIgR forms overnight. After washing, thecaptured IgM or dIgA were detected using anti-IgM or anti-IgA conjugatedto horseradish peroxidase (HRP) and colorimetric substrate TMB. Theresults demonstrate that HpIgR shows preferential binding to IgM(magenta) as well as binding to dIgA (green), whereas R/HpIgR showsgreatly reduced binding to IgM (yellow) but retains strong binding todIgA (blue).

EXAMPLE 2 ELISA Shows Detection of Immobilised dIgA Using R/HpIgR withNegligible Background

An ELISA was performed comparing the detection of immobilised dIgA usingR/HpIgR. Dilutions of purified dIgA or no dIgA (mock) were immobilisedon 96-well Nunc Immulon plates that were previously coated withanti-IgA, so that the dIgA was bound to the plate by antibody-antigeninteraction rather than passive absorption. The dIgA was detected usingR/HpIgR (“tailless”) or no pIgR (“mock”), anti-secretory component andanti-mouse HRP and TMB substrate. The results (see FIG. 9) demonstrateR/HpIgR is able to detect dIgA at the lowest concentration tested (31ng/ml) with strong signal in ELISA with negligible background.

EXAMPLE 3 ELISA Showing Effective Depletion of HAV-Specific IgM in IgMCapture

An ELISA was conducted demonstrating the detection of HAV-specific IgMin IgM capture, using serial dilutions of serum from a patient withacute HAV infection (Accurun HAV panel sample 121). The serum sample iseither untouched before dilution (untouched, purple) or substantiallydepleted of IgM using Capture-Select IgM (BAC) (red). The results (seeFIG. 12) show that this IgM depletion method reduces the level ofHAV-specific IgM in the sample by around 256-fold compared to untouchedserum.

EXAMPLE 4 ELISA Shows Detection of Hepatitis A Virus-Specific dIgACompared to IgM in an Individual Patient, with or without Depletion ofIgM

An ELISA was conducted demonstrating the detection of HAV-specific dIgAin R/HpIgR capture, using serial dilutions of serum from a patient withacute HAV infection (Accurun HAV panel sample 121). The serum sample iseither untouched before dilution (untouched, purple) or substantiallydepleted of IgM using Capture-Select IgM (BAC) (red). The results showfirstly the strong signal that is obtained demonstrating the detectionof HAV-specific dIgA, and secondly that this signal is specific for dIgAnot IgM because the IgM depletion method did not substantially reducethe level of HAV-specific reactivity compared to untouched serum, incontrast to the results shown in FIG. 12 for IgM detection.

EXAMPLE 5 R/HpIgR Capture of Antigen-Specific dIgA for the Diagnosis ofAcute HAV Infection

An ELISA was conducted demonstrating the detection of HAV-specific dIgAin R/HpIgR capture, using serial dilutions of serum from a patient withacute HAV infection (Accurun HAV panel sample 121). The serum sample iseither untouched before dilution (untouched, purple) or substantiallydepleted of IgM using Capture-Select IgM (BAC) (red). The results (seeFIG. 13) show firstly the strong signal that is obtained demonstratingthe detection of HAV-specific dIgA, and secondly that this signal isspecific for dIgA not IgM because the IgM depletion method did notsubstantially reduce the level of HAV-specific reactivity compared tountouched serum, in contrast to the results shown in FIG. 12 for IgMdetection.

An ELISA was performed demonstrating the detection of HAV-specific dIgAin R/HpIgR capture, using sera from patients with or without acute HAVinfection (Accurun HAV panel, positive (POS), low positive (LOW POS), ornegative (NEG)). The results (FIG. 14) show the strong detection ofHAV-specific dIgA in all POS samples and in one of two LOW POS samples,with minimal background reactivity in NEG samples, demonstrating theutility of R/HpIgR capture of antigen-specific dIgA for the diagnosis ofacute HAV infection.

EXAMPLE 6 R/HpIgR Capture and Utility of HpIgR Capture for Diagnosis ofAcute HEV Infection

An ELISA was conducted demonstrating the detection of hepatitis E virus(HEV)-specific dIgA in R/HpIgR capture or HpIgR capture, using sera frompatients with or without acute HEV infection. On the left of FIG. 15,the ELISA OD of individual samples is shown, demonstrating the utilityof R/HpIgR capture of antigen-specific dIgA for the diagnosis of acuteHEV infection, with lower but still significant utility of HpIgR capturefor this purpose, and negligible background reactivity in eitherexample. On the right of FIG. 15, the reactivity of serial dilutions ofeach serum sample is shown, confirming the utility of R/HpIgR captureand lower utility of HpIgR capture for diagnosis of acute HEV infection.It is likely that the lower utility of HpIgR capture in these examplesis due to the much higher overall concentration of IgM in serum versusdIgA, resulting in only a low proportion of the IgM captured by HpIgRbeing specific for HEV.

EXAMPLE 7 Detection of Antigen-Specific dIgA or IgM Illustrated UsingHEV Infected Patients

Comparison of HEV-specific dIgA versus HEV-specific IgM. Both methodsare able to detect all HEV-infected patients with strong ELISA signals(FIG. 17), compared to extremely low background for control(HEV-negative) patients in the dIgA assay, and low background in the IgMassay. Notably, some samples show higher levels of dIgA compared to IgM(sample J13, J7), while others show higher levels of IgM compared todIgA (J4, J11). This demonstrates that the dIgA and IgM responses inpatients are independent, and suggests that a combination of both IgMand dIgA detection may be useful in some desirable assay formats. FIG.17 illustrates that the dIgA assay involved essentially no backgroundwhile the commercial IgM assay which is highly optimised gives low butdetectable background.

Comparison of HEV-specific dIgA versus HEV-specific IgM is conductedusing sera that are either untouched, or substantially depleted of IgMusing Capture-Select IgM, and then serially diluted. The results (FIG.18) confirm that the IgM assay is specific for IgM, because thereactivity is ablated by IgM depletion, whereas the dIgA assay ispredominantly specific for dIgA and not IgM, because the reactivity isonly slightly affected by IgM depletion.

Comparison of HEV-specific dIgA versus HEV-specific IgM is conductedusing sera that are either untouched, or substantially depleted of IgMusing Capture-Select IgM. The results (FIG. 19) confirm that the IgMassay is specific for IgM, because the reactivity is ablated by IgMdepletion, whereas the dIgA assay is predominantly specific for dIgA andnot IgM, because the reactivity is only slightly affected by IgMdepletion.

Comparison of HEV-specific dIgA versus HEV-specific IgM is conductedusing sera that are either untouched, or substantially depleted of IgMusing Capture-Select IgM. The results (FIG. 20) confirm that the IgMassay is specific for IgM, because the reactivity is ablated by IgMdepletion, whereas the dIgA assay is predominantly specific for dIgA andnot IgM, because the reactivity is only slightly affected by IgMdepletion. The reduction in dIgA activity following IgM depletion isstatistically significant when using a paired T-test to compare samplesbefore and after depletion, but is not significant when using aMann-Whitney test to compare the overall sample sets before and afterdepletion.

EXAMPLE 8 R/HpIgR Capture and Detection of Mouse dIgA

An ELISA is conducted demonstrating that the R/HpIgR can be used in bothcapture (A) and detection (B) of mouse dimeric IgA, with negligiblebackground reactivity to monomeric (human) IgA. FIG. 21A. Dilutions ofpurified mouse IgA monoclonal antibody 3H1 (anti-HAV) or purifiedmonomeric human IgA were coated on plates and detected with R/HpIgR andanti-SC antibodies. B. R/HpIgR was coated on plates and dilutions ofpurified mouse IgA monoclonal antibody 3H1 or purified monomeric humanIgA were allowed to bind overnight, then detected with anti-mouse IgA oranti-human IgA. The binding of IgA from diverse species to human orrabbit pIgR is known in the art, and this demonstrates that the novelpIgR strategy described herein has utility for diagnosis of infection inother species.

An ELISA is conducted demonstrating that the R/HpIgR is equallyeffective for detection of mouse dimeric IgA and human dimeric IgA, withnegligible background reactivity to monomeric human IgA. FIG. 22A.Dilutions of purified mouse IgA monoclonal antibody 3H1 (anti-HAV) orpurified dimeric or monomeric human IgA were coated on plates anddetected with R/HpIgR and anti-SC antibodies.

EXAMPLE 9 Assessment of HIV Pathogenesis and Illustration of Utility ofSIgA/dIgA Ratios in HIV-Infected Subjects

A schematic of one of several typical assays (see FIG. 27) that can beused to measure the relative amount of different IgA forms in order toestimate the SIgA/dIgA ratio. In this example, the amount of dIgA ismeasured by capture of dIgA using R/HpIgR, and detection usingmonoclonal antibodies against either IgA1, or IgA2, or against both IgAsubclasses. Monomeric IgA does not bind to pIgR; SIgA does bind toR/HpIgR but with lower affinity than dIgA and can be removed by washingwith 3.5 M urea if desired. SIgA is measured in the same way but usinganti-SC antibody capture instead of R/HpIgR. The SIgA/dIgA ratio is thencalculated as a simple ratio of the assay reactivities for SIgA anddIgA.

An ELISA is conducted demonstrating the detection of highly elevatedSIgA2/dIgA2 (S/d) ratios in a proportion of HIV-infected patients,compared to the majority of HIV-infected patients and all controlsubjects (magenta). The assay cutoff for elevated SIgA/dIgA was set asthe mean plus 3 standard deviations of the SIgA/dIgA ratio among non-HIVcontrol subjects, and 7/30 HIV-infected subjects showed SIgA/dIgA ratiosabove this cutoff. Notably, the range of SIgA/dIgA ratios among normalsubjects is smaller than the range for SIgA or dIgA alone, because therole of dIgA as the precursor of SIgA provides a normalising effect foreach patient.

An ELISA is conducted demonstrating the total amount of SIgA in patientand control sera (arbitrary units). The amount of SIgA2 in normalpatients varies over an 11-fold range, but all normal controls fallwithin a cutoff of the mean plus 3 standard deviations. The amount ofSIgA2 in HIV-infected patients varies over a slightly larger range(16-fold), but only 2/30 patients are above the cutoff range (see FIG.29). Among the HIV-infected patients, those patients who demonstratedelevated SIgA2/dIgA2 ratios in FIG. 28 are indicated with red markers(diamonds at ranks 11, 16, 19, 23, 24, 28 and 30). It can be seen thatthese patients with elevated SIgA2/dIgA2 ratios are found throughoutmuch of the normal range of the total SIgA2 signal, and cannot bedistinguished from the normal controls on the basis of the total SIgA2alone. This confirms the utility of using SIgA/dIgA ratios because therole of dIgA as the precursor of SIgA provides a normalising effect foreach patient. The R/HpIgR system provides the utility for measuring thisratio.

FIG. 30 illustrates a correlation of SIgA2/dIgA2 ratio versus the immuneactivation marker, CD8+ HLA-DR+ CD38+ T-cells, in a differentHIV-infected population to that shown in FIGS. 28 and 29. While theoverall correlation is low, it is apparent that patients withSIgA2/dIgA2 ratios of >4 in this experiment have elevated levels ofimmune activation markers (p<0.0001).

FIG. 31 illustrates a correlation of SIgA1/dIgA1 ratio versus the immuneactivation marker, CD8+ HLA-DR+ CD38+ T-cells, in the same population asFIG. 30. While the overall correlation is lower again than for IgA2, itis apparent that patients with SIgA1/dIgA1 ratios of >10 in thisexperiment have elevated levels of immune activation markers (p<0.015).The lower correlation for IgA1 and higher cutoff ratio (10 versus 4) forsignificance highlights the value of specifically measuring IgA2 becauseof its predominant site of synthesis in the gut, being the tissue inwhich leakage of SIgA is likely to be clinically relevant marker of gutleakage and immune activation.

FIG. 32 illustrates a correlation of SIgA1/dIgA1 ratio versusSIgA2/dIgA2 ratio, in the same population as FIGS. 30 and 31. WhileSIgA1/dIgA1 ratios are significantly correlated with SIgA2/dIgA2, it isnotable that there are some patients with highly elevated SIgA1/dIgA1ratios and relatively low SIgA2/dIgA2 ratios. This suggests that theremay be some value in measuring IgA1, or total IgA, in addition to IgA2in the calculation of SIgA/dIgA ratios as a measure of gut leakage andimmune activation.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

Many modifications will be apparent to those skilled in the art withoutdeparting from the scope of the present invention.

TABLE 1 Summary of sequence identifiers SEQUENCE ID NO: DESCRIPTION 1Nucleotide sequence of human pIgR 2 Amino acid sequence of human pIgR 3Nucleotide sequence of rabbit pIgR 4 Amino acid sequence of rabbit pIgR5 Nucleotide sequence of chimeric human/rabbit pIgR 6 Amino acidsequence of chimeric human/rabbit pIgR 7 Nucleotide sequence ofN-terminal rabbit domain 1 chimeric human/rabbit pIgR 8 Amino acidsequence of N-terminal rabbit domain 1 chimeric human/rabbit pIgR 9Nucleotide sequence of C-terminal human domains 2-4 of chimerichuman/rabbit pIgR 10 Amino acid sequence of C-terminal human domains 2-4of chimeric human/rabbit pIgR 11 Nucleotide sequence of human pIgR withcytoplasmic domain of CD4 12 Amino acid sequence of human pIgR withcytoplasmic domain of CD4 13 Nucleotide sequence of rabbit pIgRcytoplasmic domain with cytoplasmic domain of CD4 14 Amino acid sequenceof rabbit pIgR with cytoplasmic domain of CD4 15 Nucleotide sequence ofchimeric human/rabbit pIgR with cytoplasmic domain of CD4 16 Amino acidsequence of chimeric human/rabbit pIgR with cytoplasmic domain of CD4 17Nucleotide sequence of N-terminal rabbit domain 1 of chimerichuman/rabbit pIgR with cytoplasmic domain of CD4 18 Amino acid sequenceN-terminal rabbit domain 1 of chimeric human/rabbit pIgR withcytoplasmic domain of CD4 19 Nucleotide sequence of C-terminal humandomains 2-4 of chimeric human/rabbit pIgR with cytoplasmic domain of CD420 Amino acid sequence of C-terminal human domains 2-4 of chimerichuman/rabbit pIgR with cytoplasmic domain of CD4

TABLE 2 Amino acid sub-classification Sub-classes Amino acids AcidicAspartic acid, Glutamic acid Basic Noncyclic: Arginine, Lysine; Cyclic:Histidine Charged Aspartic acid, Glutamic acid, Arginine, Lysine,Histidine Small Glycine, Serine, Alanine, Threonine, ProlinePolar/neutral Asparagine, Histidine, Glutamine, Cysteine, Serine,Threonine Polar/large Asparagine, Glutamine Hydrophobic Tyrosine,Valine, Isoleucine, Leucine, Methionine, Phenylalanine, TryptophanAromatic Tryptophan, Tyrosine, Phenylalanine Residues that Glycine andProline influence chain orientation

TABLE 3 Exemplary and Preferred Amino Acid Substitutions OriginalPreferred Residue Exemplary Substitutions Substitutions Ala Val, Leu,Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu CysSer Ser Gln Asn, His, Lys, Asn Glu Asp, Lys Asp Gly Pro Pro His Asn,Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleu Leu Leu Norleu,Ile, Val, Met, Ala, Phe Ile Lys Arg, Gln, Asn Arg Met Leu, Ile, Phe LeuPhe Leu, Val, Ile, Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp TyrTyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleu Leu

BIBLIOGRAPHY

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1. An antibody capture process comprising (i) obtaining a biologicalsample comprising antibodies, (ii) contacting the biological sample withrecombinant pIgR or a dIgA-binding variant, wherein the pIgR or variantbinds dIgA and forms a pIgR-dIgA complex.
 2. The process of claim 1,further comprising (iii) directly or indirectly assessing the level ofthe pIgR-dIgA complex or the level of a complex between pIgR-dIgA and anantigen of interest.
 3. The antibody capture process of claim 1 or claim2 wherein the pIgR or a dIgA-binding variant binds dIgA andsubstantially fails to bind IgM or wherein the pIgR or variant bindsdIgA and IgM.
 4. The antibody capture process of claims 1 to 3 whereinthe biological sample is a blood or serum sample.
 5. The antibodycapture process of any one of claims 1 to 4 wherein the biologicalsample is obtained from a subject.
 6. The antibody capture process ofclaim 5 wherein the subject is human or primate.
 7. The antibody captureprocess of claim 5 wherein the subject is a mammalian or avian animalspecies other than a primate or human species.
 8. The antibody captureprocess of any one of claims 1 to 7 wherein the pIgR is recombinantHpIgA or RpIgR.
 9. The antibody capture process of any one of claims 1to 7 wherein the recombinant pIgR or dIgA-binding variant has thetransmembrane domain and/or the cytoplasmic domain deleted.
 10. Theantibody capture process of any one of claims 1 to 9 wherein therecombinant pIgR comprises a heterologous detection or binding domain.11. The antibody capture process of any one of claims 1 to 0 wherein therecombinant pIgR or IgA-binding variant is recombinantly produced in aglycan deficient cell.
 12. The antibody capture process of any one ofclaims 1 to 11 wherein the recombinant pIgR is bound to a solid support.13. The antibody capture process of any one of claims 1 to 12 whereinthe biological sample is depleted of IgM or dIgA antibodies prior to usein the process.
 14. The process of any one of claims 2 to 13 wherein theantigen of interest is an antigen of an infectious agent or an antigenassociated with a condition of a subject that affects a mucosal surfaceor associated tissues.
 15. The process of claim 13 wherein theinfectious agent is selected from HIV, leprosy, syphilis, hepatitis,dengue virus, measles and rubella.
 16. The process of any one of claims1 to 14 process further comprising contacting the biological sample withan anti-SC binding agent or anti-SC antibody wherein the anti-SC bindingagent or anti-SC antibody binds SIgA and forms an SIgA-bindingagent/antibody complex.
 17. The process of any one of claims 1 to 14,further comprising contacting a sample comprising the pIgR-dIgA complexwith a denaturing solution to remove any SIgA from the complex andmeasuring the ratio of SIgA and dIgA in the biological sample.
 18. Anantibody capture process for determining gut wall integrity in a testsubject, the process comprising (i) obtaining a biological samplecomprising antibodies from the test subject, (ii) contacting thebiological sample with recombinant pIgR or a dIgA-binding variant,wherein the pIgR or variant binds dIgA and forms a pIgR-dIgA complex,and (iii) contacting the biological sample with a specific anti-SIgAbinding agent or anti-SC binding agent/antibody wherein the anti-SIgAbinding agent or anti-SC binding agent/antibody binds SIgA and forms anSIgA-binding agent/antibody complex, and (iv) measuring and comparingthe level of the complex formed in (ii) with the level of the complexformed in (iii), wherein the ratio of SIgA to dIgA is compared to acorresponding level or ratio from a control subject and provides ameasure of gut integrity/leakage.
 19. A process of any one of claims 16to 18 wherein the level or ratio of SIgA2/dIgA2 and/or SIgA1/dIgA1 aredetermined.
 20. An antibody capture process comprising (i) obtaining abiological sample comprising antibodies, (ii) contacting the biologicalsample with recombinant pIgR or a dIgA or IgM-binding variant, whereinthe pIgR or variant binds IgM and/or IgA and forms a pIgR-IgM and/orpIgR-dIgA complex.
 21. An antibody capture process comprising (i)obtaining a biological sample comprising antibodies, (ii) contacting thebiological sample with recombinant pIgR wherein the pIgR or variantbinds IgM and forms a pIgR-IgM complex.
 22. A process for detecting thepresence of antigen-specific dIgA in a subject, the process comprising(i) obtaining a biological sample comprising antibodies from a subject,(ii) contacting the sample with R/HpIgR and antigen and (iii) measuringthe level of antigen-specific dIgA.
 23. A process for detecting thepresence of antigen-specific IgM in a subject, the process comprising(i) obtaining a biological sample comprising antibodies from a subject,(ii) contacting the sample with a HpIgR and antigen and (iii) measuringthe level of antigen-specific IgM.
 24. A process for detecting thepresence of antigen-specific IgM and dIgA in a subject, the processcomprising (i) obtaining a biological sample comprising antibodies froma subject, (ii) contacting the sample with HpIgR and R/HpIgR and antigenand (iii) measuring the level of antigen-specific IgM and antigenspecific dIgA.
 25. A kit for assessing immune status in a subject, thekit comprising, (a) an immunographic device comprising a porous membraneoperably connected to a sample portion, a test portion, and optionally acontrol portion; and further comprising a sucker portion, portioncomprising a recombinant pIgR molecule or dIgA-binding variant thereof,a portion comprising an antigen of interest and optionally a conjugateportion; and b) instructions for using the immunographic device todetect the presence of antigen specific dIgA antibody in the sample. 26.The kit of claim 25 wherein the pIgR is HpIgR and/or R/HpIgR.
 27. Thekit of claim 25 or 26 wherein the recombinant pIgR or dIgA-bindingvariant has the transmembrane domain and/or the cytoplasmic domaindeleted.
 28. The kit of any one of claims 25 to 27 wherein therecombinant pIgR comprises a heterologous detection or binding domain.29. The kit of any one of claims 25 to 28 wherein the recombinant pIgRor IgA-binding variant is recombinantly produced in a glycan deficientcell.
 30. The kit of any one of claims 25 to 29 wherein the recombinantpIgR is bound to a solid support.
 31. The kit of any one of claims 25 to30 wherein the antigen of interest is an antigen of an infectious agentor an antigen associated with a condition of a subject that affects amucosal surface or associated tissues.
 32. The kit of claim 31 whereinthe infectious agent is selected from HIV, leprosy, syphilis, hepatitis,dengue virus, measles and rubella.
 33. The kit of any one of claims 25to 32 further comprising an anti-SIgA binding agent/antibody or anti-SCantibody, wherein the anti-SIgA binding agent/antibody or anti-SCantibody binds SIgA and forms an SIgA-binding agent/antibody complex.34. Recombinant pIgR when used for, or for use, in capturing ordetecting dIgA and/or IgM.
 35. The recombinant pIgR of claim 34, whichis R/HpIgR or HpIgR or a dIgA and/or IgM binding variant of R/HpIgR orHpIgR.